Development of Malolactic Fermentation Process Using Immobilized Whole Cells and Enzymes

Abstract

The purpose of this study was to develop a rapid and well controlled malolactic fermentation process. Six strains of malolactic organisms were screened for morphology and growth characteristics. The Leuconostoc oenos strains PSU-1 and 8-14 exhibited the most consistent growth characteristics in complex media. Cells of strain PSU-1 grown to a concentration of 10⁸ cells/mL, using a 14 L fermentor, were immobilized in a 3% K-carrageenan gel at a concentration of 10⁹ to 10¹⁰ cells/mL gel. The cells, after a 24 hour activation period, were assayed for malolactic activity in a tartrate buffer, pH 4.2, 2 mM MnSO₄ with malate added at 1 to 20 mM. The average specific activity found was 10 mM malate/hr/g dry cell with a K_m of 5.4 mM. These results were compared to the average specific activity found for free cell suspensions of 18 mM malate/hr/g dry cell and K_m of 2.0 mM. The lower rates for immobilized cells were attributed in large part to loss in cell density in the gel from 10⁹ down to 10⁷ cells/mL gel after immobilization. Purification of the malolactic enzyme from crude cell extract containing 0.8 units/mg protein has produced a crude enzyme preparation which showed a specific activity of 16.5 units/mg protein.